Synthesis and biological properties of the seven alanine-modified analogues of the marine cyclopeptide hymenamide C.

The synthesis and biological activity of the marine cyclopeptide hymenamide C(1), showing an inhibitory effect on human neutrophil elastase degranulation release, were recently described. Based on this result, it was decided to undertake a systematic structure-activity relationship study of this cyclopeptide, based on the Ala-scan technique, in order to obtain useful information for the rational design of additional analogues. The synthesis and characterization of the seven Ala modified analogues are reported and their biological and pharmacological properties are described.     

N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry

To evaluate the ability of an insect cell-free protein synthesis system to generate proper N-terminal cotranslational protein modifications such as removal of the initiating Met, N-acetylation, and N-myristoylation, several mutants were constructed using truncated human gelsolin (tGelsolin) as a model protein. Tryptic digests of these mutants were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The wild-type tGelsolin, which is an N-myristoylated protein, was found to be N-myristoylated when myristoyl-CoA was added to the in vitro translation reaction mixture. N-myristoylation did not occur on the Gly-2 to Ala mutant, in which the N-myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiating Met. Analyses of Gly-2 to His and Leu-3 to Asp mutants revealed that the amino acids at positions 2 and 3 strongly affect the susceptibility of the nascent peptide chain to removal of the initiating Met and to N-acetylation, respectively. These results suggest that N-terminal modifications occurring in the insect cell-free protein synthesis system are quite similar to those observed in the mammalian protein synthesis system. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein modifications.     

Semi-Mechanized Simultaneous Synthesis of Multiple Oligonucleotide Fragments

Abstract

A new strategy for the simultaneous synthesis of multiple oligonucleotides is described, which is based on asynchronous chain growth and use of a maximum number of support segments in each elongation. An approach to the mechanization of this strategy and applications to the preparation of gene fragments and sequentially variant probes are discussed.     

Citicoline: neuroprotective mechanisms in cerebral ischemia.

Cytidine-5'-diphosphocholine (citicoline or CDP-choline), an intermediate in the biosynthesis of phosphatidylcholine (PtdCho), has shown beneficial effects in a number of CNS injury models and pathological conditions of the brain. Citicoline improved the outcome in several phase-III clinical trials of stroke, but provided inconclusive results in recent clinical trials. The therapeutic action of citicoline is thought to be caused by stimulation of PtdCho synthesis in the injured brain, although the experimental evidence for this is limited. This review attempts to shed some light on the properties of citicoline that are responsible for its effectiveness. Our studies in transient cerebral ischemia suggest that citicoline might enhance reconstruction (synthesis) of PtdCho and sphingomyelin, but could act by inhibiting the destructive processes (activation of phospholipases). Citicoline neuroprotection may include: (i) preserving cardiolipin (an exclusive inner mitochondrial membrane component) and sphingomyelin; (ii) preserving the arachidonic acid content of PtdCho and phosphatidylethanolamine; (iii) partially restoring PtdCho levels; (iv) stimulating glutathione synthesis and glutathione reductase activity; (v) attenuating lipid peroxidation; and (vi) restoring Na(+)/K(+)-ATPase activity. These observed effects of citicoline could be explained by the attenuation of phospholipase A(2) activation. Based on these findings, a singular unifying mechanism has been hypothesized. Citicoline also provides choline for synthesis of neurotransmitter acetylcholine, stimulation of tyrosine hydroxylase activity and dopamine release.     

Making by Grinding: Mechanochemistry Boosts the Development of Halide Perovskites and Other Multinary Metal Halides

Mechanochemical synthesis has recently emerged as a promising route for the synthesis of functional lead halide perovskites as well as other (lead‐free) metal halides. Mechanochemical synthesis presents several advantages with regards to more commonly used solution‐based processes such as an inherent lower toxicity by avoiding organic solvents and a finer control over stoichiometry of the final products. The ease of implementation, either through the use of a simple mortar and pestle or with an electrically powered ball‐mill, and low amount of side products make mechanochemical synthesis appealing for upscaling the production of halide perovskites. Due to the defect tolerance of lead halide perovskites, they are ideally suited to be prepared by this solvent‐free method. However, the implementation of these semiconductors in high‐efficiency optoelectronic devices requires the transformation of synthesized powder into smooth thin films where still some hurdles remain to be cleared.     

Interleukin (IL)-17 enhances tumor necrosis factor-alpha-stimulated IL-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

Inflammatory cytokines are well known to play crucial roles in the pathogenesis of rheumatoid arthritis. Among them, interleukin (IL)-17 is a cytokine that is mainly synthesized by activated T cells and its receptors are present in osteoblasts. The synthesis of IL-6, known to stimulate osteoclastic bone resorption, is reportedly responded to bone resorptive agents such as tumor necrosis factor-alpha (TNF-alpha) in osteoblasts. It has been reported that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We previously showed that sphingosine 1-phosphate (S1-P) mediates TNF-alpha-stimulated IL-6 synthesis in these cells. In the present study, we investigated the mechanism of IL-17 underlying enhancement of IL-6 synthesis in MC3T3-E1 cells. IL-17 induced phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, significantly reduced the enhancement by IL-17 of TNF-alpha-stimulated IL-6 synthesis. IL-17 also amplified S1-P-stimulated IL-6 synthesis, and the amplification by IL-17 was suppressed by SB203580. Anisomycin, an activator of p38 MAP kinase, which alone had no effect on IL-6 level, enhanced the IL-6 synthesis stimulated by TNF-alpha. SB203580 and PD169316 inhibited the amplification by anisomycin of the TNF-alpha-induced IL-6 synthesis. Taken together, our results strongly suggest that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.     

Synthesis of of beta-amyloid precursor peptide and presenilin segments.

It seems likely that the beta-amyloid precursor protein (APP) and the presenilins (PS-1/2) play important roles in the development of Alzheimer's disease (AD). Attempts to mimic the biochemical actions of these proteins are often made by the application of fragments of these proteins. However, the synthesis of these segments by conventional methods of peptide synthesis is problematic. We have synthesized several C-terminal fragments of APP and PS-1/2 by solid-phase synthesis through combination of automatic and manual methods of synthesis. This permits solution of the 'difficult sequences' in the solid-phase synthesis of these peptides. Some details of the syntheses of nine segments are presented in this paper.     

Solid-Phase Cross-Linking (SPCL): a new tool for protein structure studies

A wide range of chemical reagents are available to study the protein-protein interactions or protein structures. After reaction with such chemicals, covalently modified proteins are digested, resulting in shorter peptides that are analyzed by mass spectrometry (MS). Used especially when NMR of X-ray data are lacking, this methodology requires the identification of modified species carrying relevant information, among the unmodified peptides. To overcome the drawbacks of existing methods, we propose a more direct strategy relying on the synthesis of solid-supported cleavable monofunctional reagents and cross-linkers that react with proteins and that selectively release, after protein digestion and washings, the modified peptide fragments ready for MS analysis. Using this Solid-Phase Cross-Linking (SPCL) strategy, only modified sequences are analyzed and consistent data can be easily obtained since the signals of interest are not masked or suppressed by over-represented unmodified materials.     

Juvenile hormone acid methyl transferase is a key regulatory enzyme for juvenile hormone synthesis in the Eri silkworm, Samia cynthica ricini

During the period of adult emergence in the Eri silkworm, Samia cynthia ricini, the corpora allata (CA) are apparently reactivated in females, but not males. This creates a significant sexual dimorphism in juvenile hormone (JH) synthesis by CA. To determine the underlying molecular mechanisms in this process, we cloned cDNAs of two enzymes involved in the JH synthesis pathway: 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and juvenile hormone acid methyl transferase (JHAMT). Both Samcri-HMGR and -JHAMT mRNAs were detected in CA almost exclusively. However, their expression patterns were different from each other. During the period of adult emergence, Samcri-HMGR was expressed in CA at a constantly high level suggesting it plays little role for the regulation of JH synthesis. In contrast, the patterns of both Samcri-JHAMT mRNA level and enzyme activity were closely correlated with the patterns of JH synthesis, CA reactivation, and sexual dimorphism of JH synthesis. In addition, JHAMT mRNA levels were paralleled JH synthesis in the fifth-instar larvae of S. cynthia ricini and the pharate adults of the silkworm Bombyx mori. We infer from these results that JHAMT is a key regulatory enzyme for JH synthesis in the Eri silkworm.     

Fmoc‐based peptide thioester synthesis with self‐purifying effect: heading to native chemical ligation in parallel formats

The chemical synthesis of proteins has facilitated functional studies of proteins due to the site‐specific incorporation of post‐translational modifications, labels, and non‐proteinogenic amino acids. Moreover, native chemical ligation provides facile access to proteins by chemical means. However, the application of the native chemical ligation reaction in the synthesis of parallel formats such as protein arrays has been complicated because of the often cumbersome and time‐consuming synthesis of the required peptide thioesters. An Fmoc‐based peptide thioester synthesis with self‐purification on the sulfonamide ‘safety‐catch’ linker widens this bottleneck because HPLC purification can be avoided. The method is based on an on‐resin cyclization–thiolysis reaction sequence. A macrocyclization via the N‐terminus of the full‐length peptide followed by a thiolytic C‐terminal ring opening allows selective detachment of the truncation products and the full‐length peptide. A brief overview of the chemical aspects of this method is provided including the optimization steps and the automation process. Furthermore, the application of the cyclization–thiolysis approach combined with the native chemical ligation reaction in the parallel synthesis of a library of 16 SH3‐domain variants of SHO1 in yeast is described, demonstrating the value of this new technique for the chemical synthesis of protein arrays. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Optimized selective N-methylation of peptides on solid support.

Peptides containing N(alpha)-methylamino acids exhibit interesting therapeutic profiles and are increasingly recognized as potentially useful therapeutics. Unfortunately, their synthesis is hampered by the high price and nonavailability of many N(alpha)-methylamino acids. An efficient and practical three-step procedure for selective N-methylation of peptides on solid support is described. The procedure was based on the well known solid-phase N-methylation of N(alpha)-arylsulfonyl peptides, which was improved by using dimethylsulfate and the less expensive DBU as base. Every step of the procedure, amine activation by an o-nitrobenzenesulfonyl group, selective N-methylation and removal of the sulfonamide group, was optimized in respect of time and economy. The described optimized three-step procedure is performed in 35 min without solvent changes, instead of 3 h. Tripeptides (Fmoc-Phe-MeXaa-Leu-OH) containing N-methylated common amino acids were also prepared using the optimized procedure to demonstrate its compatibility with these amino acids. The described procedure allows an efficient synthesis of N(alpha)-methylamino acid containing peptides in a very short time using Fmoc solid-phase peptide synthesis.     

Amino acid deletion products resulting from incomplete deprotection of the Boc group from Npi-benzyloxymethylhistidine residues during solid-phase peptide synthesis.

In peptide synthesis, the use of N(alpha)-tert-butyloxycarbonyl-N(pi)-benzyloxymethylhistidine [Boc-His(pi-Bom)] raises the problem of the Bom group generating formaldehyde during the hydrogen fluoride (HF) cleavage reaction. This can lead to modification of the functional groups on amino acids in the peptide chain. Besides this side reaction, the failure of N(alpha)-Boc deprotection from the His(pi-Bom) residue occurs during TFA treatment for the standard solid-phase peptide synthesis (SPPS) even in the case of a non 'difficult sequence'. This gives amino acid deletion products generated at the N-terminus of the His(pi-Bom) residues. Reviewing the removability of the Boc group on amino acid derivatives showed that the group on the His(pi-Bom) residue was much more resistant under the deprotecting conditions than expected. To circumvent this problem, special precautions, i.e. prolonged deprotection steps and/or increased concentrations of TFA, should be taken for a successful SPPS.     

Hydrophobic benzyl amines as supports for liquid‐phase C‐terminal amidated peptide synthesis: application to the preparation of ABT‐510

C‐terminal amidation is one of the most common modification of peptides and frequently found in bioactive peptides. However, the C‐terminal modification must be creative, because current chemical synthetic techniques of peptides are dominated by the use of C‐terminal protecting supports. Therefore, it must be carried out after the removal of such supports, complicating reaction work‐up and product isolation. In this context, hydrophobic benzyl amines were successfully added to the growing toolbox of soluble tag‐assisted liquid‐phase peptide synthesis as supports, leading to the total synthesis of ABT‐510 ( 2 ). Although an ethyl amide‐forming type was used in the present work, different types of hydrophobic benzyl amines could also be simply designed and prepared through versatile reductive aminations in one step. The standard acidic treatment used in the final deprotection step for peptide synthesis gave the desired C‐terminal secondary amidated peptide with no epimerization. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Constitutive collagenase-1 synthesis through MAPK pathways is mediated, in part, by endogenous IL-1alpha during fibrotic repair in corneal stroma

Collagenase-1 is a protease expressed by active fibroblasts that is involved in remodeling of the extracellular matrix (ECM). In this study, we characterize the intracellular signaling mechanism of collagenase-1 production by IL-1alpha in subcultured normal fibroblasts (NF) from uninjured normal corneas, compared to that in repair wound fibroblasts (WF). In NF, collagenase-1 was induced specifically after the exogenous addition of IL-1alpha via activation of ERK and p38MAPK. Collagenase-1 expression was strongly suppressed upon treatment with either a MEK or p38MAPK inhibitor. In contrast, repair WF constitutively synthesized both IL-1alpha and collagenase-1. Combined treatment with both mitogen-activated protein kinase (MAPK) inhibitors dramatically reduced collagenase-1 synthesis, while individual MEK1 or p38 inhibitors weakly modulated the collagenase-1 level. The results indicate that both pathways are crucial in the regulation of collagenase-1 synthesis. Furthermore, an IL-1alpha receptor antagonist (IL-1ra) could not abolish constitutive collagenase-1 synthesis, even at high doses, suggesting that other cytokines/factors are additionally involved in this process. We propose that induction of collagenase-1 by IL-1alpha in both WF and NF depends on a unique combination of cell type-specific signaling pathways.     

1,4-diazepine-2,5-dione ring formation during solid phase synthesis of peptides containing aspartic acid beta-benzyl ester.

The Fmoc-based SPPS of H-Xaa-Asp(OBzl)-Yaa-Gly-NH(2) sequences results in side reactions yielding not only aspartimide peptides and piperidide derivatives, but also 1,4-diazepine-2,5-dione-peptides. Evidence is presented to show that the 1,4-diazepine-2,5-dione derivative is formed from the aspartimide peptide. The rate of this ring transformation depends primarily on the tendency to aspartimide and piperidide formation, which is influenced by the nature of the amino acid following the aspartic acid beta-benzyl ester (Xaa). However the bulkiness of the amino acid side chain preceeding the aspartic acid beta-benzyl ester (Yaa) is also important. Under certain conditions the 1,4-diazepine-2,5-dione peptide derivative may even be formed dominantly, which is a highly undesirable side reaction in peptide synthesis, but which provides a new way for the synthesis of diazepine peptide derivatives with targeted biological or pharmacological activity.     

EFFECT OF NUTRIENT DEPRIVATION AND RESUPPLY ON METABOLITES AND ENZYMES RELATED TO CARBON ALLOCATION IN GRACILARIA TENUISTIPITATA (RHODOPHYTA)1

The starch content of red algae normally increases during nitrogen limitation. Based on this we hypothesized that nutrient deprivation would result in an increased activity of starch‐synthesizing enzymes and a decrease in the activity of starch‐degrading enzymes, with the opposite scenario when nutrients were sufficient. We therefore examined the effect of the nutrient status of Gracilaria tenuistipitata Chang et Xia on the content of starch and floridoside and on the activity of enzymes involved in the allocation of carbon into starch, floridoside, and agar; floridoside phosphate synthase and α‐galactosidase involved in synthesis and degradation of floridoside; starch synthase and starch phosphorylase involved in the metabolism of starch; uridine 5′‐diphosphate (UDP)‐glucose pyrophosphorylase; adenosine 5′‐diphosphate‐glucose pyrophosphorylase; UDP‐glucose 4‐epimerase; and phosphoglucomutase. During the period of nutrient limitation the starch and floridoside content increased, as did dry weight and C/N ratio, whereas growth rate and protein content decreased. A general decrease in the enzyme activities during nutrient limitation was also observed, indicating a decrease in overall cellular metabolism. The addition of nutrients caused an increase in enzyme activities and a decrease in the contents of starch and floridoside. Of the enzymes examined, only the activity of UDP‐glucose pyrophosphorylase increased during nutrient limitation and decreased abruptly after nutrient addition. This implies a regulatory role for this enzyme in the supply of UDP‐glucose for starch synthesis. It also supports our suggestion that UDP‐glucose is the substrate for starch synthesis in red algae. This assertion is further strengthened by the observation that of the potential starch synthases only the UDP‐glucose starch synthase could support the observed rate of starch synthesis.     

Epac1‐induced cellular proliferation in prostate cancer cells is mediated by B‐Raf/ERK and mTOR signaling cascades

cAMP‐dependent, PKA‐independent effects on cell proliferation are mediated by cAMP binding to EPAC and activation of Rap signaling. In this report, we employed the analogue 8‐CPT‐2‐O‐Me‐cAMP to study binding to EPAC and subsequent activation of B‐Raf/ERK and mTOR signaling in human cancer cells. This compound significantly stimulated DNA synthesis, protein synthesis, and cellular proliferation of human 1‐LN prostate cancer cells. By study of phosphorylation‐dependent activation, we demonstrate that EPAC‐mediated cellular effects require activation of the B‐Raf/ERK and mTOR signaling cascades. RNAi directed against EPAC gene expression as well as inhibitors of ERK, PI 3‐kinase, and mTOR were employed to further demonstrate the role of these pathways in regulating prostate cancer cell proliferation. These studies were then extended to several other human prostate cancer cell lines and melanoma cells with comparable results. We conclude that B‐Raf/ERK and mTOR signaling play an essential role in cAMP‐dependent, but PKA‐independent, proliferation of cancer cells. J. Cell. Biochem. 108: 998–1011, 2009. © 2009 Wiley‐Liss, Inc.     

High‐Temperature Shock Enabled Nanomanufacturing for Energy‐Related Applications

Functional nanomaterials are playing a crucial role in the emerging field of energy‐related devices. Recently, as a novel synthesis method, high‐temperature shock (HTS), which is rapid, low cost, eco‐friendly, universal, scalable, and controllable, has provided a promising option for the rational design and synthesis of various high‐quality nanomaterials. In this report, the HTS technique, including the equipment setup and operating principle, is systematically introduced, and recent progress in the synthesis of nanomaterials for energy storage and conversion applications using this HTS method is summarized. The growth mechanisms of nanoparticles and carbonaceous nanomaterials are thoroughly discussed, followed by the summary of the characteristic advantages of the HTS strategy. A series of nanomaterials prepared by the HTS method, including carbon‐based films, metal nanoparticles and compound nanoparticles, show high performance in the diverse applications of storage energy batteries, highly active catalysts, and smart energy devices. Finally, the future perspectives and directions of HTS in nanomanufacturing for broader applications are presented.     

Preparation of tri(ethylene glycol) grafted core‐shell type polymer support for solid‐phase peptide synthesis

A core‐shell type polymer support for solid‐phase peptide synthesis has been developed for high coupling efficiency of peptides and versatile applications such as on‐bead bioassays. Although various kinds of polymer supports have been developed, they have their own drawbacks including poor accessibility of reagents and incompatibility in aqueous solution. In this paper, we prepared hydrophilic tri(ethylene glycol) (TEG) grafted core‐shell type polymer supports (TEG SURE) for efficient solid‐phase peptide synthesis and on‐bead bioassays. TEG SURE was prepared by grafting TEG derivative on the surface of AM PS resin via biphasic diffusion control method and subsequent acetylation of amine groups which are located at the core region of AM PS resin. The performance of TEG SURE was evaluated by synthesizing several peptides. Three points can be highlighted: (1) easy control of loading level of TEG, (2) improved efficiency of peptide synthesis compared with the conventional resins, and (3) applicability of on‐bead bioassays.     

OPP Labeling Enables Total Protein Synthesis Quantification in CHO Production Cell Lines at the Single‐Cell Level

Accurate measurement of global and specific protein synthesis rates is becoming increasingly important, especially in the context of biotechnological applications such as process modeling or selection of production cell clones. While quantification of total protein translation across whole cell populations is easily achieved, methods that are capable of tracking population dynamics at the single‐cell level are still lacking. To address this need, we apply O‐propargyl‐puromycin (OPP) labeling to assess total protein synthesis in single recombinant Chinese hamster ovary (CHO) cells by flow cytometry. Thereby we demonstrate that global protein translation rates slightly increase with progression through the cell cycle during exponential growth. Stable CHO cell lines producing recombinant protein display similar levels of total protein synthesis as their parental CHO host cell line. Global protein translation does not correlate with intracellular product content of three model proteins, but the host cell line with high transient productivity has a higher OPP signal. This indicates that production cell lines with increased overall protein synthesis capacity can be identified by our method at the single‐cell level. In conclusion, OPP‐labeling allows rapid and reproducible assessment of global protein synthesis in single CHO cells, and can be multiplexed with DNA staining or any type of immunolabeling of specific proteins or markers for organelles.